ISSN 10619348, Journal of Analytical Chemistry, 2015, Vol. 70, No. 1, pp. 32–38. © Pleiades Publishing, Ltd., 2015.

Original Russian Text © N.E. Kuz’mina, S.V. Moiseev, V.I. Krylov, V.A. Yashkir, V.A. Merkulov, 2015, published in Zhurnal Analiticheskoi Khimii, 2015, Vol. 70, No. 1, pp. 30–36.

ARTICLES

Determination of the Average Molecular Weight of Hydroxyethyl

Starches by DiffusionOrdered NMR Spectroscopy
N. E. Kuz’mina, S. V. Moiseev*, V. I. Krylov, V. A. Yashkir, and V. A. Merkulov
Scientific Center for Expertise of Medical Products, Ministry of Health of the Russian Federation,
Petrovskii bul’var 8, Moscow, 127051 Russia
*email: MoiseevSV@expmed.ru
Received October 9, 2013; in final form, February 17, 2014

Abstract—A correlation equation is obtained for the evaluation of the average molecular weight (MW) of
hydroxyethyl starches from the selfdiffusion coefficient D measured using diffusionordered NMR spectros
copy. Correlation equations MW = cD–α are compared for hydroxyethyl starches, dextrans, and pullulans. It
has been demonstrated that different MWs correspond to constant D values of uncharged polysaccharides
with the similar type of nonvalent interactions and different degrees of branching.

Keywords: diffusionordered NMR spectroscopy, average molecular weight of polymers, selfdiffusion coef
ficient, hydroxyethyl starches
DOI: 10.1134/S1061934815010086

INTRODUCTION branched polymer amylopectin consisting of αD

Hydroxyethyl starches (HES) are efficient colloid
plasma substituents, which are widely used in clinical
practice for the prevention and treatment of hypov
olemia in surgery, traumatology, combustiology, and
intensive therapy over the last 50 years [1–3]. The
molecular structure of HES represents a polydisperse
glucopyranose residues modified with hydroxyethyl
groups (Scheme). The linear section of the HES mac
romolecules contains α(1 → 4) bonds, branching are
formed mostly by α(1 → 6) bonds [4, 5]. The side
branches of the macromolecule contain from 15 to
45 glucose residues.

OH
O O
OH O O
OH
O O
O O
O OH
O O HO
HO O O
HO O O OH
O
HO OH O
OH
OH HO O

OH
Fragment of the molecular structure of HES.

The important characteristics of HES affecting
their pharmacokinetic properties are the average
molecular weight; molar substitution (MS). which is
the ratio of the number of hydroxyethyl groups to the
total number of glucose molecules; and the ratio
C2/C6, which characterizes the ratio of the number of
hydroxyethyl groups at the second carbon atom to the
number of hydroxyethyl groups at the sixth carbon
atom in the glucopyranose cycle [6–8]. MW deter
mines the time of the HES presence in the blood flow.
HES macromolecules and fragments of their enzy
matic hydrolysis penetrating through the kidney bar
rier (45–60 kDa) are rapidly eliminated from the body
[7]. Hydroxyethylation slows down the enzymatic

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 DETERMINATION OF THE AVERAGE MOLECULAR WEIGHT
hydrolysis of the HES molecules, thus extending the
time of circulation of the preparation in the blood flow
[8]. A similar effect is made by the high C2/C6 ratio:
the higher the C2/C6 ratio, the slower the decomposi
tion of HES [6–8].
The determination of the quantitative characteris
tics of HES (MW, MS, and C2/C6) is a rather complex
and laborious problem; in the pharmacopeia analysis
it is solved using a complex of chromatographic meth
ods: gelpermeation chromatography (GPC) (MW
determination), and GC and HPLC (determination
of MS and C2/C6) [9, 10]. The application of chro
matographic methods to the determination of MS and
C2/C6 is based on the destruction of the analyzed
HES. Therefore, the development of a nondestructive
rapid method that ensures the quick and reliable esti
mation of all quantitative HES properties using one
sample with the minimal sample preparation is a prob
lem of current importance. For the development of
such a method mostly NMR spectroscopy is used. The
quantitative determination of MS and sites of HES
substitution was performed based on the data of 1H
and 13C NMR spectra [4, 11–16]. Methods of tradi
tional NMR spectroscopy are inapplicable to the
determination of the MWs of polymers. Diffusion
ordered NMR spectroscopy (DOSY), which ensures
the measurement of the diffusion of various molecular
objects (molecules, macromolecules, molecular com
plexes, and supramolecular systems) under the impact
of a magnetic field gradient [17–19], seems to be the
most promising method for this purpose. The DOSY
method possesses high potential in the studies of the
molecular weights of polymers [20–23], including
polysaccharides [24–28]. In practice [26–28], the
MWs of polysaccharides within the frameworks of the
DOSY method were evaluated using the correlation
equation proposed by the authors of [24] for the
dependence of the MW of polysaccharides on the self
diffusion coefficient D, which is numerically equal to
the mean square shift of a molecule during the time of
diffusion td [29]. This equation was obtained on the
basis of the analysis of linear and lowbranched
polysaccharides (dextrans and pullulans). It is known
that the topology of a polymer substantially affects its
diffusion, because branched molecules always have
smaller hydrodynamic radii than their linear ana
logues [30]; therefore the use of the correlation equa
tion for the quantitative evaluation of the MW of HES
presented in [24] can lead to incorrect results.
The aim of the present work was to study the effect
of branching on the D value of polysaccharides and the
evaluation of a correlation relationship between the
MW of HES and D taking into account the peculiari
ties of their steric structures.
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 70
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EXPERIMENTAL
We used HES standard samples produced by the
American Polymer Standard Corporation (United
1
States) with the average molecular weights Мр 5.5,
8.9, 16.1, 52.8, 80.0,121.1, 153.6, 176.1, 201.0, 219.3,
330.2, 374.0, 396.9, 468.7, and 1004.0 kDa (HETA
6K, HETA 9K, HETA 18K, HETA 60K, HETA 88K,
HETA 130K, HETA 160K, HETA 175K, HETA
230K, HETA 250K, HETA 337K, HETA 380K,
HETA 460K, HETA 600K, and HETA 1300K,
respectively). The 1H NMR and 1HDOSY spectra
were recorded on an Agilent DD2 NMR System 600
NMRspectrometer with a 5 mm inverse multinuclear
sensor equipped with a gradient coil at 300 K. The
analyzed samples (5 mg) were dissolved in 0.5 mL of
D2O (Cambridge Isotope Laboratories Inc.). The
solutions obtained were diluted to the concentration
1 mg/mL and transferred into Shigemi NMR tubes
with a glass plunger for the minimization of the effects
of convection and the heterogeneity of pulses of the
magnetic field gradient. For measurements of the self
diffusion coefficient, a DBPPSTE (DOSY Bipolar
Pulse Pair Stimulated Echo) sequence was used. Dif
fusion attenuation curves were obtained at a sequential
15step linear increase of the amplitude of the mag
netic field gradient pulse in the range from 1.82 to
53 G/cm at fixed values of diffusion time Δ (285 ms),
δpulse gradient pulse duration (2.0 ms), and relaxation
time d1 (5 s).
The results were mathematically processed using
the DISCRETE method (Discrete Sum of Exponen
tial Decays). Correlation equation was derived using
the MS Excel 2007 software.
RESULTS AND DISCUSSION
HES is a polydisperse polymer; its MW is an aver
age value and depends on the mode of averaging. In
polymer chemistry, number average (Mn) and weight
average (Мw) molecular weights are distinguished. Mn
is obtained by averaging by the number of macromol
ecules in a polymer; Мw is obtained by averaging by the
weight of macromolecules in a polymer. The Mw value
is sensitive to highmolecular fractions, while Mn is
sensitive to lowmolecular weight fractions of polydis
perse polymers; therefore, for the characteristic of the
average MW of HES we used an averaged parameter
representing molecular weight in peak (Мp), deter
mined by chromatographic methods. For narrowdis
persed samples Мp = (Mw × Mn)0.5 [31]. The correlation
relationship between the MW and D values is
described by a power function [24, 27, 32, 33]:
D = cMWα,
1M is the molecular weight of a peak in its maximum in the
p
determination of the average molecular weight of a polymer by
exclusion gelpermeation chromatography.
No. 1 2015